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Background: EPHOS-B is a multi-centre randomized trial designed to investigate whether anti-HER2 therapy given 11 days pre-surgery inhibited proliferation and/or increased apoptosis in HER2-positive early breast cancer. Significant differences in Ki67 response (≥30% change between baseline and surgery) were observed between treatment groups and control, although no significant increases in apoptosis were seen [1]. We report here 5-year outcomes and their association with peri-operative response, including post-hoc exploratory analyses on stromal tumour infiltrating lymphocytes (TILs). Methods: Patients were randomized (1:2:2) to no treatment (control), trastuzumab or lapatinib (Part-1), and (1:1:2) to control, trastuzumab, or lapatinib and trastuzumab combination (Part-2). Relapse free survival (RFS) is defined as the time from randomisation to local, regional, or distant tumour recurrence or death from any cause, with second primary cancers censored. Five-year RFS rates were estimated by Kaplan-Meier across treatment and peri-operative response groups, and compared by Log-Rank tests. Central scoring of stromal tumour infiltrating lymphocytes (TILs) on scanned H&E baseline and surgery slides was conducted post-hoc according to the International TILs Working Group. Baseline TILs (bTILS) were explored across treatment groups, and associated with trial outcomes. Changes in TILS in patients without pathological regression were correlated with Ki67 changes and RFS. Results: Overall, 257 patients were randomized (Part-1: control 22, trastuzumab 57, lapatinib 51; Part-2: control 29, trastuzumab 32, trastuzumab+lapatinib 66). Central pathology review identified six patients achieving complete pathological response (pCR), and 13 RCB1 (as per the Residual Cancer Burden score), all but two in the combination group. After 6 years median follow-up, 28 women (11%) had recurrence, and 19 (7%) died; no differences between groups were found (Table). No recurrences were observed amongst pCR patients; only 1 local recurrence observed amongst RCB1. Patients with Ki67 falls ≥50% had better RFS than those with modest falls or no falls (Table). In patients with no evidence of disease regression at surgery, those with higher TILs (≥20%) at surgery had better RFS than those with lower TILs. Conclusions: Early response (pCR/RCB1 or Ki67 reductions >50%) after 11 days pre-operative anti-HER2 dual therapy identifies cancers dependent on the HER2 pathway and provides a strategy for individualising treatment, including de-escalation of therapy. [1] Bundred N, Cameron D, Amstrong A et al (2016) Effects of perioperative lapatinib and trastuzumab, alone and in combination, in early HER2+ breast cancer − the UK EPHOS-B trial (CRUK/08/002) European Journal of Cancer;57:S1-S8. Table - Relapse-free survival by treatment and by peri-operative responseN randomisedNRFS event5-year RFS95%CILog-rank p-valueRandomised treatmentTrastuzumab (P1)577 (12.3%)88%76-94%T vs L p=0.75Lapatinib (P1)515 (9.8%)90%77-96%L vs C p=0.41Control (P1)221 (4.6%)95%92-99%Trastuzumab (P2)327 (21.9%)87%69-95%T vs T+L p=0.048Combination (P2)665 (7.6%)92%83-97%T+L vs C p=0.64Control (P2)293 (10.3%)90%71-97%%Ki67 change:>50% decrease722 (2.8%)99%90-100%0.002>10% decrease7717 (22.1%)80%69-88%20%502 (4%)96%85-99Surgery TILS (only RCB2/3)20%653 (4.6%)95%86-98Change in TILS (only RCB2/3)No significant increase15221 (13.8%)88%81-920.16Increase >20%352 (5.7%)94%79-99 Citation Format: Nigel Bundred, Nuria Porta, Adrian Murray Brunt, Angela Cramer, Andrew Hanby, Abeer Shaaban, Emad Rakha, Anne Armstrong, Ramsey Cutress, David Dodwell, Marie Emson, Abigail Evans, Sue Hartup, Kieran Horgan, Stuart McIntosh, Jay Naik, Samkaran Narayanan, Jane Oii, Anthony Skene, David Cameron, Judith Bliss. Combined peri-operative lapatinib and trastuzumab in early HER2-positive breast cancer - Long term results of the randomized UK EPHOS-B trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-08.

Purpose:The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation.Patients and Methods:STAKT was a two-stage, double-blind, randomized, placebo-controlled, “window-of-opportunity” study in patients with newly diagnosed ER+ invasive breast cancer. Stage 1 assessed capivasertib 480 mg b.i.d. (recommended monotherapy dose) and placebo, and stage 2 assessed capivasertib 360 and 240 mg b.i.d. Primary endpoints were changes from baseline in AKT pathway markers pPRAS40, pGSK3β, and proliferation protein Ki67. Pharmacologic and pharmacodynamic properties were analyzed from blood sampling, and tolerability by adverse-event monitoring.Results:After 4.5 days' exposure, capivasertib 480 mg b.i.d. (n = 17) produced significant decreases from baseline versus placebo (n = 11) in pGSK3β (H-score absolute change: −55.3, P = 0.006) and pPRAS40 (−83.8, P < 0.0001), and a decrease in Ki67 (absolute change in percentage positive nuclei: −9.6%, P = 0.031). Significant changes also occurred in secondary signaling biomarker pS6 (−42.3, P = 0.004), while pAKT (and nuclear FOXO3a) also increased in accordance with capivasertib's mechanism (pAKT: 81.3, P = 0.005). At doses of 360 mg b.i.d. (n = 5) and 240 mg b.i.d. (n = 6), changes in primary and secondary biomarkers were also observed, albeit of smaller magnitude. Biomarker modulation was dose and concentration dependent, and no new safety signals were evident.Conclusions:Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.

Supplementary Data from Proliferation and AKT Activity Biomarker Analyses after Capivasertib (AZD5363) Treatment of Patients with ER+ Invasive Breast Cancer (STAKT)

Purpose Changes occur in the expression of oestrogen-regulated and proliferation-associated genes in oestrogen receptor (ER)-positive breast tumours during the menstrual cycle. We investigated if Oncotype® DX recurrence score (RS), Prosigna® (ROR) and EndoPredict® (EP/EPclin) prognostic tests, which include some of these genes, vary according to the time in the menstrual cycle when they are measured. Methods Pairs of test scores were derived from 30 ER-positive/human epidermal growth factor receptor-2-negative tumours sampled at two different points of the menstrual cycle. Menstrual cycle windows were prospectively defined as either W1 (days 1–6 and 27–35; low oestrogen and low progesterone) or W2 (days 7–26; high oestrogen and high or low progesterone). Results The invasion module score of RS was lower (− 10.9%; p = 0.098), whereas the ER (+ 16.6%; p = 0.046) and proliferation (+ 7.3%; p = 0.13) module scores were higher in W2. PGR expression was significantly increased in W2 (+ 81.4%; p = 0.0029). Despite this, mean scores were not significantly different between W1 and W2 for any of the tests and the two measurements showed high correlation (r = 0.72–0.93). However, variability between the two measurements led to tumours being assigned to different risk categories in the following proportion of cases: RS 22.7%, ROR 27.3%, EP 13.6% and EPclin 13.6%. Conclusion There are significant changes during the menstrual cycle in the expression of some of the genes and gene module scores comprising the RS, ROR and EP/EPclin scores. These did not affect any of the prognostic scores in a systematic fashion, but there was substantial variability in paired measurements.

Background: Prognosis in older patients with breast cancer (BC) is worse compared to younger patients. No robust and specific tool to predict the risk of recurrence (TTR) for women aged 70 and over is likely due to the lack of representation of this group in the data from clinical trials. In the POETIC trial, of estrogen receptor-positive (ER+) and mainly human epidermal growth factor receptor 2 negative (HER2-) BC (88%), peri-operative aromatase inhibitor (POAI) did not improve treatment outcome, but patients with low baseline Ki67 value (Ki67B) or low POAI-induced Ki67 value (Ki67_2wk) had good outcome with standard of care therapy (usually adjuvant endocrine therapy (adjET) with the addition of chemotherapy as clinically indicated). In this study, we sought to develop a multi-parametric algorithm, named Ki67Cal, by integrating Ki67_2wk value with tumour characteristics to predict TTR for patients > 70 years old (yr) with early ER+HER2- BC treated with adjET only. Methods: Within POETIC, 39% (n=1744) of the randomised patients (n = 4480) were >70 years old. There were 813 patients aged > 70yr, with ER+HER2- BC, randomised to POAI treatment and treated with adjET only. A power calculation indicated that 811 such patients were sufficient to develop a prediction model that minimized overfitting, allowed up to 8 predictors, for predicting 5-years TTR with a median follow-up of 5.24 years and an overall event rate per 1000 person-years = 0.027, and provided an anticipated performance in terms of model fit R2 = 0.08 (Riley et al. BMJ 2020). A three-fold cross-validation approach was applied; an optimal list of features was selected in the training set (n = 538, events = 70); the agreement between expected and observed outcomes from the algorithm on the validation set (n = 275, events = 37) was evaluated by calibration plot. Multivariable Ridge Cox Regression model of significant parameters was built on the dataset merging training and validation datasets (n = 813) for precise estimates of the coefficients of parameters. A subset of post-POAI samples (n = 99) was gene expression profiled with Nanostring to allow pseudo-Oncotype, pseudo-EndoPredict, and RUO-Prosigna scores calculated (Buus et al. npj Breast cancer 2021). The risk groups classified by the Ki67Cal and gene-expression assays (GEP) were compared. Results: Within this cohort, the 5-year TTR was 34.5% (C.I. 24.9-47.9) for those with a high Ki67_2wk (>=10%) and 12.3% (C.I. 9.1-16.7) in those with a high Ki67B that was suppressed to Ki672wk < 10%. The significant features were Ki67_2wk, sampling type (core vs. excision) at surgery, and pathological variables (tumour size, grade, and nodal status) for the final Ki67Cal algorithm. Stratifying patients into five groups (quintiles) by Ki67Cal identified 60% of patients with TTR of < 5% at 5yrs, and 20% of patients with TTR of > 30% at 5yrs. As an exploratory analysis, the risk groups by Ki67Cal and GEP were compared (Table 1). To date, these assays are optimized to be used on untreated ER+HER2- samples; there were fairly good agreements between the high-risk group defined by Ki67Cal with pseudo-EndoPredict and RUO-Prosigna respectively, and low-risk groups by Ki67Cal with Prosigna probably because Prosigna scores are driven by proliferation score. Conclusion: The relatively poor outcome of patients >70yrs in POETIC emphasizes the need for prognostic tools that identify patients who may be treated with endocrine therapy alone or conversely should be considered for additional therapy. Ki67Cal provides a simple tool that identified very low-risk and high-risk patients in 80% of patients with ER+HER2- BC. Table 1: Comparison of the risk groups defined by Ki67Cal algorithm with the three commonly used gene-expression assays (pseudo-EndoPredict, pseudo-Oncotype and RUO-Prosigna) applied on the post-peri-operative aromatase inhibitor samples. Citation Format: Maggie Chon U Cheang, Monisha Dewan, Lucy Kilburn, Gabriele Morani, Lila Zabaglo, Kally Sidhu, Holly Tovey, Xixuan Zhu, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Alistair Ring, Nicholas Turner, Judith Bliss, Mitch Dowsett. Multi-parametric algorithm integrating on-treatment Ki67 value and standard clinicopathological variables to predict risk of recurrences for women > 70 years old with early ER+HER2- tumours in POETIC trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-07.

Background: POETIC was a phase III clinical trial, with patients randomised 2:1 to 2-week perioperative aromatase inhibitor (POAI) vs control for postmenopausal women with oestrogen receptor positive (ER+) early breast cancer (BC) (Smith et al., Lancet Oncology 2020). Our previous study on POETIC trial patients with ER+ human epidermal growth factor receptor 2 positive (HER2+) BC suggested both HER2 enriched subtype (HER2-E) and immune enrichment pre-POAI (baseline, B) are main drivers of poor early response to POAI (Bergamino et al., 2022). However, some patients with HER2-E or immune enriched BC at B still showed good response to POAI. In this study, we aim to further investigate a sub-cohort of ER+ HER2+ BC from the POETIC trial, including a subset of aforementioned HER2-E tumours, to further explore the multi-modal molecular characteristics of the tumours resistant to POAI. Methods: Proliferation rate was assessed as percentage of cancer cells stained by Ki67. Patient POAI response was determined by Ki67 reduction at 2 weeks of treatment. A sub-cohort of 37 patients were selected based on response and classified as poor responders (PR, reduction < 30%, n=18), good responders (GR, reduction > 90%, n=11) and good responders with HER2-E BC at B (GR, reduction > 65%, n=8). Paired B and post-POAI (surgery, S) samples were taken from each patient of the sub-cohort. Multiplex immunofluorescence (mIF) was performed on these samples, measuring the immune cell densities in stroma and tumour compartments using five biomarkers: CD3 (all T cells), CD20 (B cells), CD68 (Macrophages), FOXP3 (regulatory T cells), and CD3 FOXP3 co-expression. The samples were also profiled using Breast Cancer 360TM (NanoString, BC360), covering the expressions of 758 genes and 46 biological signatures. Wilcoxon test, hierarchical clustering and spearman correlation test were performed to compare the tumour characteristics of GR and PR. Results: In this study, two B and four S samples were not achievable for mIF experiments due to low tumour content. At B (n = 35), among the five mIF biomarker measurements in stroma and tumour, only the stromal CD3 density was significantly different between GR (median = 0.0013) and PR (median = 0.0003, p = 0.041). In GR, HER2-E BC at B were separated into immune-high and immune-low groups with mIF biomarkers at B; the immune-high group was more likely to change into luminal subtypes post-POAI, while the immune-low group remained HER2-E. After POAI, the density changes in five mIF biomarkers in stroma and CD68 in tumour were all significantly higher in PR than GR (Table 1, n of paired samples = 62). The BC360 signatures of BC p53 (p < 0.001), BC proliferation (p < 0.001), LumB (p < 0.001) and HER2-E correlation coefficients (p < 0.001) were significantly downregulated in GR after POAI, while LumA correlation coefficients (p < 0.001) were notably increased. Conclusions: Our results suggest that for this sub-cohort, increased stromal immune response is associated with poor response to 2-week POAI in ER+ HER2+ early BC. HER2-E GR display visible immune heterogeneity at B. Lower-risk BC characteristics were exhibited in GR after the 2-week treatment. Further integrating mIF imaging data and additional digital spatial profiling are ongoing to reveal additional characteristics of ER+ HER2+ BC and tumour microenvironment predicting POAI resistance. Table 1: List of medians of log2 fold changes in mIF biomarker densities between GR and PR among the 62 paired samples, and Wilcoxon test p-values. Citation Format: Xixuan Zhu, Hui Xiao, Elena López-Knowles, Milana A. Bergamino Sirvén, Anastasia Alataki, Perry Maxwell, Holly Tovey, Lucy Kilburn, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Katherine A Hoadley, Roberto Salgado, Judith Bliss, Nicholas Turner, Manuel Salto-Tellez, Gene Schuster, Mitch Dowsett, Maggie Chon U Cheang. Deconstructing the molecular characteristics of ER+ HER2+ early breast cancer in the POETIC trial using multiplex immunofluorescence and gene expression profiles [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-08.

Around 25% of women undergoing Axillary Clearance (ANC) develop lymphedema (LE). Intervention with a compression garment is recommended to prevent LE but no randomised evidence exists to support this strategy.A randomised trial tested standard management versus application of graduated compression garments (20-24 mmHg) to affected arm, for 1 year. Women with node positive breast cancer (n = 1300) undergoing ANC consented to arm volume measurements and those developing a 4-9% relative arm volume increase (RAVI) (subclinical LE) within 9 months post-surgery were randomised. Primary outcome was proportion of patients developing LE (RAVI 10%) by 24-months in each group. Secondary endpoints included Quality of life in each group.In total 143 patients were randomised (74 no sleeve: 69 compression sleeve) between October 2010 and November 2015. The lymphoedema rate at 24 months in the 'no sleeve' group was at 41%, similar to the 'sleeve' group (30%: p = 0.32). Thirtytwo patients randomised to the 'no sleeve' group had a sleeve applied within 24 months. Body Mass Index (BMI) at randomisation predicted LE at any time point HR 1.04 (CI 1.01-1.08; p = 0.01). Patients with obesity (BMI 30) had higher rates of LE in both groups (46%) compared to those with BMI 30 (24%). No difference between patients was found in either group in changes in QoL. Compression sleeves applied after development of LE improved QoL scores (FACT-B p = 0.007:TOI p = 0.042).Early intervention with External Compression garments does not prevent clinical LE, particularly in women with a high BMI 30. The use of prophylactic garments in subclinical LE (RAVI 9%) is unwarranted.

Background: oestrogen receptor positive/ human epidermal growth factor receptor positive (ER+/HER2+) breast cancers (BCs) are less responsive to endocrine therapy than ER+/HER2- tumours. Mechanisms underpinning the differential behaviour of ER+HER2+ tumours are poorly characterised. Our aim was to identify biomarkers of response to 2 weeks’ presurgical AI treatment in ER+/HER2+ BCs. Methods: all available ER+/HER2+ BC baseline tumours (n=342) in the POETIC trial were gene expression profiled using BC360™ (NanoString) covering intrinsic subtypes and 46 key biological signatures. Early response to AI was assessed by changes in Ki67 expression and residual Ki67 at 2 weeks (Ki67 2wk). Time-To-Recurrence (TTR) was estimated using Kaplan-Meier methods and Cox models adjusted for standard clinicopathological variables. New molecular subgroups (MS) were identified using consensus clustering. Findings: HER2-enriched (HER2-E) subtype BCs (44.7% of the total) showed poorer Ki67 response and higher Ki67 2wk (p2wk. Five new MS that were associated with differential response to AI were identified. HER2-E had significantly poorer TTR compared to Luminal BCs (HR 2.55, 95% CI 1.14–5.69; p=0.0222). The new MS were independent predictors of TTR, adding significant value beyond intrinsic subtypes. Interpretation: our results show HER2-E as a standardised biomarker associated with poor response to AI and worse outcome in ER+/HER2+. HRD, TP53 mutational score and immune-tumour tolerance are predictive biomarkers for poor response to AI. Lastly, novel MS identify additional non-HER2-E tumours not responding to AI with an increased risk of relapse. Funding: Cancer Research UK (CRUK/07/015).

Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

BackgroundEndocrine therapy reduces breast cancer mortality by 40%, but resistance remains a major clinical problem. In this study, we sought to investigate the impact of aromatase inhibitor (AI) therapy on gene expression and identify gene modules representing key biological pathways that relate to early AI therapy resistance.MethodsGlobal gene expression was measured on pairs of core-cut biopsies taken at baseline and at surgery from 254 patients with ER-positive primary breast cancer randomised to receive 2-week presurgical AI (n = 198) or no presurgical treatment (controln = 56) from the POETIC trial. Data from the AI group was adjusted to eliminate artefactual process-related changes identified in the control group. The response was assessed by changes in the proliferation marker, Ki67.ResultsHigh baselineESR1expression associated with better AI response in HER2+ tumours but not HER2− tumours. In HER2− tumours, baseline expression of 48 genes associated with poor antiproliferative response (p PERPandYWHAQ, the two most significant, and the transcription co-regulators (SAP130,HDAC4, andNCOA7) which were among the top 16 most significant. Baseline gene signature scores measuring cell proliferation, growth factor signalling (ERBB2-GS, RET/GDNF-GS, andIGF-1-GS), and immune activity (STAT1-GS) were significantly higher in poor AI responders. Two weeks of AI caused downregulation of genes involved in cell proliferation and ER signalling, as expected. Signature scores of E2F activation and TP53 dysfunction after 2-week AI were associated with poor AI response in both HER2− and HER2+ patients.ConclusionsThere is a high degree of heterogeneity in adaptive mechanisms after as little as 2-week AI therapy; however, all appear to converge on cell cycle regulation. Our data support the evaluation of whether an E2F signatures after short-term exposure to AI may identify those patients most likely to benefit from the early addition of CDK4/6 inhibitors.Trial registrationISRCTN,ISRCTN63882543, registered on 18 December 2007.

The major changes in hormone levels that occur through the menstrual cycle have been postulated to affect the expression of hormone-regulated and proliferation-associated genes (PAGs) in premenopausal ER+ breast cancer. Whilst previous studies have demonstrated differences in gene expression, here, we investigated if there are within patient changes in the expression of oestrogen- and progesterone-regulated genes (ERGs and PRGs) and PAGs in ER+ breast cancer during the menstrual cycle. Samples from 96 patients in two independent prospective studies of the effect of menstrual cycle on ER+ breast cancer were used. Plasma hormone measurements were used to assign tumours to one of three pre-defined menstrual cycle windows: W1 (days 27–35 and 1–6; low oestradiol and low progesterone), W2 (days 7–16; high oestradiol and low progesterone) and W3 (days 17–26; intermediate oestradiol and high progesterone). RNA expression of 50 genes, including 27 ERGs, 11 putative PRGs and seven PAGs was measured. The AvERG (geomean of PGR, GREB1, TFF1 and PDZK1) was used as a composite measure of ERG expression and showed significant changes between the three windows of the menstrual cycle increasing over 2.2-fold between W1 and W2 and decreasing between W2 and W3 and between W3 and W1. Proliferation gene expression also varied significantly, following the same pattern of changes as ERG expression, but the changes were of lower magnitude (1.4-fold increase between W1 and W2). Significant changes in the expression of eight individual ERGs, including GREB1, PGR and TFF1, and two PAGs were observed between W1 and either W2 or W3 with all genes showing higher levels in W2 or W3 (1.3–2.4-fold; FDR 0.016–0.05). The AvProg, a composite measure of PRG expression, increased significantly (1.5-fold) in W3 compared to W1 or W2 but no significant changes were observed for individual PRGs. In conclusion, we observed significant changes in ERG, PRG and PAG expression in ER+ breast tumours during the menstrual cycle that may affect the assessment and interpretation of prominent biomarkers (e.g. PgR) and commonly used multigene prognostic signatures in premenopausal ER+ breast cancer.

Background: \ud Preoperative and perioperative aromatase inhibitor (POAI) therapy has the potential to improve outcomes in women with operable oestrogen receptor-positive primary breast cancer. It has also been suggested that tumour Ki67 values after 2 weeks (Ki672W) of POAI predicts individual patient outcome better than baseline Ki67 (Ki67B). The POETIC trial aimed to test these two hypotheses.\ud \ud Methods: \ud POETIC was an open-label, multicentre, parallel-group, randomised, phase 3 trial (done in 130 UK hospitals) in which postmenopausal women aged at least 50 years with WHO performance status 0–1 and hormone receptor-positive, operable breast cancer were randomly assigned (2:1) to POAI (letrozole 2·5 mg per day orally or anastrozole 1 mg per day orally) for 14 days before and following surgery or no POAI (control). Adjuvant treatment was given as per UK standard local practice. Randomisation was done centrally by computer-generated permuted block method (variable block size of six or nine) and was stratified by hospital. Treatment allocation was not masked. The primary endpoint was time to recurrence. A key second objective explored association between Ki67 (dichotomised at 10%) and disease outcomes. The primary analysis for clinical endpoints was by modified intention to treat (excluding patients who withdrew consent). For Ki67 biomarker association and endpoint analysis, the evaluable population included all randomly assigned patients who had paired Ki67 values available. This study is registered with ClinicalTrials.gov, NCT02338310; the European Clinical Trials database, EudraCT2007-003877-21; and the ISRCTN registry, ISRCTN63882543. Recruitment is complete and long-term follow-up is ongoing.\ud \ud Findings: \ud Between Oct 13, 2008, and April 16, 2014, 4480 women were recruited and randomly assigned to POAI (n=2976) or control (n=1504). On Feb 6, 2018, median follow-up was 62·9 months (IQR 58·1–74·1). 434 (10%) of 4480 women had a breast cancer recurrence (280 [9%] POAI; 154 [10%] control), hazard ratio 0·92 (95% CI 0·75–1·12); p=0·40 with the proportion free from breast cancer recurrence at 5 years of 91·0% (95% CI 89·9–92·0) for patients in the POAI group and 90·4% (88·7–91·9) in the control group. Within the POAI-treated HER2-negative subpopulation, 5-year recurrence risk in women with low Ki67B and Ki672W (low–low) was 4·3% (95% CI 2·9–6·3), 8·4% (6·8–10·5) with high Ki67B and low Ki672W (high–low) and 21·5% (17·1–27·0) with high Ki67B and Ki672W (high–high). Within the POAI-treated HER2-positive subpopulation, 5-year recurrence risk in the low–low group was 10·1% (95% CI 3·2–31·3), 7·7% (3·4–17·5) in the high–low group, and 15·7% (10·1–24·4) in the high–high group. The most commonly reported grade 3 adverse events were hot flushes (20 [1%] of 2801 patients in the POAI group vs six [

This abstract was withdrawn by the authors. Citation Format: Bundred NJ, Foden P, Riches K, Morris J, Evans A, Todd C, Bramley M, Skene A, Watterson D, Purushotham A, Keeley V. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-11-05.

Introduction Lymphoedema develops after surgery in 30% patients. In a prospective, multi-centre UK study, we used a validated perometer arm measurement to determine 1) the factors predicting lymphoedema development and 2) the effect of a lymphoedema diagnosis on QoL and survival. Methods Participants (n = 1100) undergoing axillary clearance at 9 centres underwent arm volume measurements pre and post-surgery by perometry, and completed FACT-B+4 and Lymphoedema checklist questionnaires. Relative arm volume increase (RAVI) of >10% diagnosed lymphoedema. Predictors of lymphoedema development were determined using logistic regression, and changes in QoL were assessed using Generalised Estimating Equation (GEE) analyses. Results Median patient age was 56 (range 22 to 90) years; 78% received radiotherapy and 65% chemotherapy. Lymphoedema was detected in 21.4% of women by perometry and 24.4% underwent sleeve application by 24 months. Initial decreases in QoL scores post-surgery were greater in patients with lymphoedema and took longer to return to baseline values (FACT-B p=0.014, TOI p=0.008, ARM subscale p RAVI at 1 month (p25-≤30 and >30, p=0.05), ER status (p=0.05) and number of positive nodes (p Local recurrence was 1.7%. One hundred and twenty-nine patients had died of breast cancer (n=88) or had distant recurrence (n=41) across the study. Lymphoedema (RAVI≥10%) by 9 months was an independent predictor of post 9 months distant disease-free cancer survival (Table). Distant Disease Free Survival (DDFS) after Lymphoedema diagnosis Single variableMultivariableVariable (between 3 and 9 months)nHazard ratio (HR) (95% CI)p-valueHR (95% CI)p-valueRAVI ≥ 10% Yes: 105 vs No9552.30 (1.39-3.81)0.0011.73 (1.01-2.96)0.047ER Status Negative: 1719532.94 (1.92-4.49) Conclusions Lymphoedema is associated with lasting quality of life deficits and early distant relapse. Women at low risk of lymphoedema (75%) can be reassured using the scoring model. Early Arm measurements at 1 month post-surgery a useful measurement time to determine lymphoedema risk and enable patients to be reassured (75%) or plan for lymphoedema monitoring. Citation Format: Bundred NJ, Foden P, Riches K, Morris J, Evans A, Todd C, Bramley M, Skene A, Purushotham A, Keeley V. Prediction model for lymphoedema, and effect of Lymphoedema diagnosis on quality of life (QoL) and distant recurrence from breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-07-01.

Background: Capivasertib (AZD5363), an AKT1,2,3 inhibitor, significantly improved progression-free and overall survival when added to paclitaxel in triple negative breast cancer (BC) patients (Schmid et al. ASCO 2018). We have previously reported in STAKT, robust target inhibition at 480mg BD versus placebo, including significant decreases in the primary biomarkers (PBs) - Ki67, pPRAS40 & pGSK3β - in primary BCs (Robertson et al. SABCS 2017). We now report the dose- and exposure-response relationship of capivasertib and the correlation between primary and secondary (pAKT, pS6, nuclear FOXO3a) tumor biomarkers. Design: STAKT was a two-stage, double blind, randomized, placebo controlled 'window-of-opportunity' trial in newly diagnosed ER+ BC patients. Stage 1 assessed capivasertib at a dose of 480mg BD p.o. versus placebo. Stage 2 assessed capivasertib at two lower doses 360mg and 240mg BD. Tumor biopsies were taken prior to 1st dose and after 4.5 days of dosing. Evaluable patients (who required pre-defined minimum baseline PD values for PBs) included placebo (n=11), capivasertib at 480mg (n=17), 360mg (n=5) and 240mg (n=6). Blood samples for pharmacokinetic (PK) studies were scheduled at pre-dose; 2, 4, optional 6 & 8 hrs post first dose on Day 1; ˜2-4 h post last dose on Day 5 (before biopsy). The % change from baseline for PBs were evaluated against the following exposure variables (placebo=0): i) Dose, ii) Observed Cmax Day 1 (˜2h post-dose), iii) Observed plasma concentration on Day 5, iv) Model-predicted plasma concentration Day 5 at time of biopsy, and v) Model-predicted AUC on Day 5. Spearman correlation coefficient measured the strength and direction of association between biomarkers. Results: · Significant mean reductions in % change from baseline were observed for the PBs pGSK3β (-39%; p · Dose-response relationships for individual % change from baseline could be described by an Emax model for all PBs. Overall, the correlation to PK exposure (observed or predicted) was similar to the correlation to dose. · Correlation coefficient analyses between biomarkers at capivasertib 480mg BD identified- i) Positive correlations for pGSK3β with Ki67 (ρ = 0.52, p-value < 0.05) & with pS6 (ρ = 0.54, p-value Conclusions · Capivasertib caused dose- and concentration- dependent effects on biomarkers after only 4.5 days. · Significant changes in the PBs were demonstrated at 480 mg BD. Biomarker changes was observed at 360mg and 240mg BD, but statistical analysis was limited by the small sample size at lower doses. · Correlation between a number of tumor biomarkers (relative changes) were identified for capivasertib 480mg BD. Citation Format: Gee J, Coleman RE, Cheung KL, Evans A, Holcombe C, Skene A, Rea D, Ahmed S, Jahan A, Horgan K, Rauchhaus P, Littleford R, Finlay P, Cheung A, Cullberg M, de Bruin E, Foxley A, Koulai L, Pass M, Schiavon G, Rugman P, Deb R, Robertson JFR. Dose- and exposure-response relationship and biomarker correlation analysis in breast tumors from patients treated with capivasertib, an AKT inhibitor, in the STAKT randomized, placebo controlled pre-surgical study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-12-01.

Background Lymphoedema develops after axillary clearance (ANC) in 25% of patients. This prospective, multi-centre study compared multi-frequency bioimpedance spectroscopy (BIS) with arm volume measurement to: (1) determine which test has better diagnostic accuracy, (2) identify factors predicting development of lymphoedema, and its effect on quality-of-life. Methods Participants (N = 1100) underwent measurements pre and post-ANC surgery for breast cancer. Relative arm volume increase (RAVI) of >10% diagnosed lymphoedema. Predictors of lymphoedema were determined using logistic regression. Optimal diagnostic method was assessed using diagnostic accuracy. Quality-of-life was assessed using the FACT B + 4 questionnaire. Results Lymphoedema was diagnosed in 22.8% women using RAVI > 10%, 45.6% using BIS criteria, while 24.5% underwent compression sleeve application by 24 months. BMI > 30 was an independent factor for both development (p = 0.005) and progression (p = 0.015) of lymphoedema. RAVI at 1 month, BMI > 30 and number of involved nodes contributed to a novel scoring model to predict lymphoedema by 36 months. Larger decreases in QoL scores post-surgery occurred in lymphoedema patients (p Conclusions RAVI measurement was the best diagnostic tool for lymphoedema. BIS alone is not appropriate for lymphoedema screening or diagnosis. BMI > 30 predicted lymphoedema diagnosis and progression.

Purpose: The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation. Patients and Methods: STAKT was a two-stage, double-blind, randomized, placebo-controlled, “window-of-opportunity” study in patients with newly diagnosed ER+ invasive breast cancer. Stage 1 assessed capivasertib 480 mg b.i.d. (recommended monotherapy dose) and placebo, and stage 2 assessed capivasertib 360 and 240 mg b.i.d. Primary endpoints were changes from baseline in AKT pathway markers pPRAS40, pGSK3β, and proliferation protein Ki67. Pharmacologic and pharmacodynamic properties were analyzed from blood sampling, and tolerability by adverse-event monitoring. Results: After 4.5 days' exposure, capivasertib 480 mg b.i.d. (n = 17) produced significant decreases from baseline versus placebo (n = 11) in pGSK3β (H-score absolute change: −55.3, P = 0.006) and pPRAS40 (−83.8, P < 0.0001), and a decrease in Ki67 (absolute change in percentage positive nuclei: −9.6%, P = 0.031). Significant changes also occurred in secondary signaling biomarker pS6 (−42.3, P = 0.004), while pAKT (and nuclear FOXO3a) also increased in accordance with capivasertib's mechanism (pAKT: 81.3, P = 0.005). At doses of 360 mg b.i.d. (n = 5) and 240 mg b.i.d. (n = 6), changes in primary and secondary biomarkers were also observed, albeit of smaller magnitude. Biomarker modulation was dose and concentration dependent, and no new safety signals were evident. Conclusions: Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.

Lymphoedema develops after axillary clearance (ANC) in 25% of patients. This prospective, multi-centre study compared multi-frequency bioimpedance spectroscopy (BIS) with arm volume measurement to: (1) determine which test has better diagnostic accuracy, (2) identify factors predicting development of lymphoedema, and its effect on quality-of-life.Participants (N = 1100) underwent measurements pre and post-ANC surgery for breast cancer. Relative arm volume increase (RAVI) of10% diagnosed lymphoedema. Predictors of lymphoedema were determined using logistic regression. Optimal diagnostic method was assessed using diagnostic accuracy. Quality-of-life was assessed using the FACT B + 4 questionnaire.Lymphoedema was diagnosed in 22.8% women using RAVI 10%, 45.6% using BIS criteria, while 24.5% underwent compression sleeve application by 24 months. BMI 30 was an independent factor for both development (p = 0.005) and progression (p = 0.015) of lymphoedema. RAVI at 1 month, BMI 30 and number of involved nodes contributed to a novel scoring model to predict lymphoedema by 36 months. Larger decreases in QoL scores post-surgery occurred in lymphoedema patients (p 0.001). Progression to moderate lymphoedema occurred in 15% patients after sleeve application.RAVI measurement was the best diagnostic tool for lymphoedema. BIS alone is not appropriate for lymphoedema screening or diagnosis. BMI 30 predicted lymphoedema diagnosis and progression.

The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation.STAKT was a two-stage, double-blind, randomized, placebo-controlled, "window-of-opportunity" study in patients with newly diagnosed ERAfter 4.5 days' exposure, capivasertib 480 mg b.i.d. (Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.

Introduction: Detection of circulating tumor DNA (ctDNA) presents a strategy to identify Molecular Residual Disease (MRD) in patients with breast cancer. Tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen lead time between ctDNA detection and relapse. We present results from a highly sensitive personalized sequencing approach for ctDNA detection of MRD based on multiple patient specific mutations. Methods: 22 early breast cancer patients (12 hormone receptor positive HER2 negative (HR+HER2-), 7 HER2+ and 3 triple negative breast cancer (TNBC)) enrolled in the ChemoNEAR sample collection study were included. Tumor DNA from FFPE samples was Whole Exome Sequenced to identify patient specific mutations and design personalized Residual Disease and Recurrence (RaDaRTM) multiplex PCR assays. Cell free DNA was extracted from 147 plasma samples (median volume 4ml, range 0.5-5ml) and sequenced with RaDaR assays, with 10-61 variants (median 41) per panel, to 100,000x per locus. A matched single timepoint buffy coat was sequenced to identify confounding CHIP mutations. A proprietary algorithm was used to identify ctDNA. Tumor Sequencing of multiple biopsy timepoints was carried out for 14 patients (mean 2.8 samples per patient) and clonal populations estimated with Pyclone. For clusters of greater than 10 mutations, RaDaR panels were supplemented with additional variants for clonal tracking. Results: At a median follow-up of 24.6 months post-surgery, MRD was identified in 100% (17/17) of relapsed patients, and in none of the 54 time points in the 5 patients that did not relapse (p=0.0002, Log rank test). Detection of ctDNA levels ranged from 7.4 parts per million (ppm), equivalent to Allele Frequency (AF) of 0.0007%, to 13,195ppm (1.3%) (median 625ppm and 0.06% AF). Median lead-time from ctDNA detection to clinical relapse in patients with extracranial disease relapse was 12.89 months (range 3.72-26.04). In three patients with brain only relapse, ctDNA was detected prior to relapse in all patients (3/3, 100%) albeit with a reduced lead time over clinical relapse (3.85, 4.21 and 5.65 months), which was not previously achievable with single mutation dPCR MRD-detection assays. In 8/14 patients with multiple tumor samples sequenced, multiple clones (mean 3.4 clones/patient) were identified, with heterogenous polyclonal relapse in 4/8 patients, and a single clone detectable in 4/8 patients. Conclusions: In a retrospective, multi-center, proof-of-principle study of early stage breast cancer patients with personalized sequencing assays, ctDNA-detected MRD associates with relapse free survival and long lead time over clinical relapse. Sequencing based ctDNA testing can detect patients with brain-only relapses, with increased sensitivity over first generation dPCR-based ctDNA assays. Citation Format: Rosalind J. Cutts, Maria Coakley, Isaac Garcia-Murillas, Lara Ulrich, Karen Howarth, Warren Emmett, Malcolm Perry, Pete Ellis, Charlene Knape, Stephen R. Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Mitch Dowsett, Ian E. Smith, Nicholas C. Turner. Molecular residual disease detection in early stage breast cancer with a personalized sequencing approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 536.

BACKGROUND Gene expression (expr) analyses are increasingly used for characterising the pharmacodynamic response of primary BC. This includes assessing ER+ BC's dependence on estrogen (E) by measuring gene expr changes after AI-treatment. However, differences in tissue sampling and other preanalytic procedures between samples taken at diagnosis (D) and surgery (S), may lead to systematic artifactual changes that are falsely ascribed to the intervention. To identify genes whose expr is truly affected by AI, we measured global gene expr changes from paired core-cut biopsies at D and S from patients in the POETIC presurgical window trial. METHODS In POETIC, 4486 postmenopausal women with primary ER+ BC were randomised 2:1 to receive perioperative AI (2 weeks pre + 2 weeks post surgery, termed Tr) or no perioperative treatment (termed Con), allowing gene expr changes to be compared between Tr and Con. RNA was extracted from paired RNA-later stored core-cuts of 56 Con and 157 Tr patients and arrayed on Illumina whole genome expr BeadChips. Raw data was extracted, transformed, normalised and batch-corrected. Probes not detected (p>0.01) in >=25% of samples were discarded. Impact of AI on genes was evaluated based on difference of the expr mean changes (log2(S/D)) of the Tr and Con samples. RESULTS In the Con group, expr of 73 genes significantly changed (FDR 615 genes were down-regulated and 472 up-regulated in Tr but not Con. The majority of down-regulated genes were cell cycle or proliferation-associated or E-regulated, including ESR1, PDZK1, GREB1, HSPB1. Functional mapping showed changes in the regulation of cyclins and cyclin dependent kinases impacting on G1/S and G2/M. Of note, up-regulated genes included CDK6 (target for CDK4/6 inhibitors) and CCND2, involved in G1/S checkpoint regulation; SNAI2, TGFB3, TGFBR2, associated with tumour invasion and metastasis; and other genes involved in aryl hydrocarbon receptor, Glioblastoma Multiforme, HIPPO and p53 signalling. CONCLUSION Expr of certain genes is altered by processes involved in presurgical window studies. In the absence of a Con group, these may be wrongly ascribed to an experimental intervention or wrongly considered as unaffected by the intervention (eg MYC in this study). Down-regulation of E-responsive and proliferation genes was an expected response to AI but increased expr of genes such as SNAI2, CCND2 and CDK6 indicates immediate tumour re-wiring and provides mechanistic support for benefit from combination therapy with a CDK4/6 inhibitor. Citation Format: Gao Q, López-Knowles E, Cheang MCU, Morden J, Martin L-A, Sidhu K, Evans D, Martins V, Dodson A, Skene A, Holcombe C, Mallon E, Abigail E, Bliss J, Robertson J, Smith I, Dowsett M. True effect of aromatase inhibitor (AI) treatment on global gene expression (expr) changes in postmenopausal ER+ breast cancer (BC) patients: A POETIC study (CRUK/07/015) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-02.

Background Higher levels of the proliferation marker Ki67 at breast cancer (BC) diagnosis are increasingly recognised to indicate poorer prognosis. Change in ΔKi67 in response to endocrine treatment reflects response. The precise relationship between Ki67 and other clinico-path factors and how associations are affected by short exposure to aromatase inhibitor (AI) has been unclear. Methods POETIC was a UK-wide, phase III, randomised trial which tested perioperative use of AI (anastrozole (A), letrozole (L)) in postmenopausal women with early BC (Dowsett JNCI Monogr 2011). Ki67 was measured in a single central lab at diagnosis (B=baseline) and 2 weeks later at surgery (S) allowing in vivo assessment of AI sensitivity. POETIC recruited 4483 women from 130 UK centres. Paired biopsies were available for 96%. Relationship between Ki67 and clinico-path factors is described by summary statistics (median) and independent associations explored in multivariable linear regression models (MVM). Analyses of Ki67 at S and ΔKi67 (reduction) were adjusted for B Ki67 and surgical sample type. Results Absolute Ki67 (B) was associated with each classic prognostic factor. Factors affecting ΔKi67 in control pts were Ki67 (B), grade (B) and surgical sample type (core cut 4.1%; excision 17.7% p Baseline Ki67 %Ki67 change (%) with 2 weeks AIVariableNMedianln(Ki67) P value (MVM)NMedianln (Ki67(S)/Ki67 (B)) P Value (MVM)All patients391315.2 252877.6 PgR (local) 0.01 5cm17416.9 11474.1 Histological type (B) Summary POETIC provides the largest multi-centre series of women in whom centrally assessed Ki67 has been correlated with classic clinico-path factors and impact of short term AI exposure explored. Choice of AI and surgical sample type are both dictated by participating site and their inter-relationship requires further review. Whether the greater suppression of Ki67 following L, a drug associated with better E2 suppression and aromatisation than A has clinical consequences is beyond the scope of this work. Relationships found (e.g. sample type, grade) are critical for interpretation of studies using Ki67 for prognosis and ΔKi67 as a pharmacodynamic response marker. Citation Format: Bliss JM, Morden J, Evans A, Holcombe C, Horgan K, Mallon E, Raghavan V, Skene A, Dodson A, Hills M, Detre S, Zabaglo L, Graf M, Banerji J, Gillman A, Robertson J, Dowsett M, Smith I, On Behalf of the POETIC Trialists. Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-01.

This abstract was withdrawn by the authors.

Background Experimental evidence (Fisher et al, 1989) & a small clinical trial (IMPACT) respectively suggested peri-operative endocrine therapy (ET) may improve long-term disease-related outcome in patients undergoing primary surgery for ER positive (ER+) breast cancer (BC) & that tumor Ki67 levels after 2 weeks of peri-operative aromatase inhibitor (POAI) therapy might offer an effective way of predicting outcome & the need for additional adjuvant treatment. POETIC (*Peri-Operative Endocrine Therapy - Individualising Care) is a phase III randomized controlled trial designed to test these hypotheses & provide data to determine whether 2 week Ki67 improves prediction beyond that by baseline Ki67 of the group who have a higher risk of relapse in the first years after diagnosis in spite of best current standard of care. Patients & methods Postmenopausal patients with ER+ BC were randomised 2:1 to either, POAI (centre choice: letrozole 2.5mg or anastrozole 1mg daily) for 14 days prior to & 14 days following surgery or no POAI (Control). Randomization was stratified by treating center; adjuvant treatment was per UK routine practice. Tissue samples were collected at baseline & surgery (FFPE) for blinded Ki67 testing. Primary endpoint was Time to Recurrence (TTR: time from randomization to loco-regional or distant recurrence or BC death). A secondary endpoint was Ki67 at baseline & after 2 weeks of AI. Results Between 2008 & 2014, 4480 patients (2976 AI, 1504 Control) were randomized from 130 UK centers. Median age was 67 (IQR 62-75), 18% had grade 3 tumors, 39% were node positive and 61% had tumor size>2cm. For adjuvant ET 314 patients (7.2%) received tamoxifen (Tam), 3695 (84.6%) an AI, 251 (5.7%) Tam changing to AI and 109 (2.5%) changing from AI to Tam. On 8 August 2017, median follow-up was 60.7 months (IQR 49.5 to 72.2). 408/4480 (9.1%) patients have had a TTR event; 263 (8.8%) allocated to POAI compared to 145 (9.6%) controls: HR=0.91 (95%CI: 0.74, 1.12) Log-rank p=0.37. Adjusted HR=0.91 (95%CI: 0.74, 1.11). The relationship of Ki67 (baseline & after 2 weeks) with TTR in both the POAI & control groups will be presented for the overall ER+ population & HER2 defined sub-groups. Discussion There was no significant evidence that four weeks of POAI improved TTR compared with no POAI. POETIC will provide definitive evidence on the role of 2 week POAI-treated Ki67 to inform future practice & trials in terms of the potential to identify a group of patients for whom current standard of care appears insufficient in the few years post diagnosis. Citation Format: Robertson JFR, Dowsett M, Bliss JM, Morden JP, Wilcox M, Evans A, Holcombe C, Horgan K, Kirwan C, Mallon E, Sibbering M, Skene A, Vidya R, Cheang M, Banerji J, Kilburn L, Dodson A, Smith I. Peri-operative aromatase inhibitor treatment in determining or predicting longterm outcome in early breast cancer – The POETIC* Trial (CRUK/07/015) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-03.

To investigate the impact of sampling methodology on gene expression data from primary estrogen receptor–positive (ER+) breast cancer biopsies, global gene expression was measured in core-cut biopsies at baseline and surgery from patients randomly assigned to receive either two weeks of presurgical aromatase inhibitor (AI; n = 157) or no presurgical treatment (n = 56). Those genes most markedly altered in the AI group (eg, FOS, DUSP1, RGS1, FOSB) were similarly altered in the no treatment group; some widely investigated genes that were apparently unaffected in the AI group (eg, MYC) were counter-altered in the control group, masking actual AI-dependent changes. In the absence of a control group, these artefactual changes would likely lead to the most affected genes being the erroneous focus of research. The findings are likely relevant to all archival collections of ER+ breast cancer.

Aims 1. To determine the variability of mutational profiles and sub-clonality in core-cut biopsies from ER+ BC and the impact of 2-weeks’ AI therapy on these. 2. To identify mutations or patterns of mutations associated with poor anti-proliferative response to AI treatment. Background DNA alterations may lead to de novo and acquired resistance to medical therapies including AIs. Assessing this requires single time-point or sequential sampling usually with core-cuts but there is little information on their ability to represent mutational profiles or sub-clonal structure. We studied this in paired biopsies from ER+ BC primaries in 60 selected postmenopausal patients from the Peri-Operative Endocrine Therapy for Individualising Care (POETIC) trial (CRUK/07/015) before and after 2-weeks’ non-steroidal AI or no AI (randomised 2:1). Methods DNA was extracted from RNAlater-preserved diagnostic and surgical 14-gauge core-cut samples and peripheral blood from 20 no AI (Control) and 40 AI-treated patients (15 poor and 25 good Ki67-responders [PR and GR, respectively]). Patients with low ER+ BC or unsuppressed estradiol on treatment were not considered. Exome sequencing (Illumina HiSeq 2000) achieved >60% coverage across the exome at 15x depth. Variants were validated by re-sequencing (median >100x) together with 79 genes of interest curated from COSMIC and selected publications. Statistically significant genes (SMGs) were determined using MuSiC. Sub-clonality was analysed by SciClone. Results Good quality exomes were obtained on 102 samples including 44 pairs (control n=14; PR n=10; GR n=20). There were 5684 mutations (including 3616 missense and 1322 silent) affecting 3261 genes. SMGs in this series were PIK3CA (35.3%), TP53 (27.5%), CDH1 (13.8%), HEATR7B2 (8.8%), GATA3 (5.9%), CENPF (5.9%), MAP3K1 (5.9%), MAP2K4 (4.9%), HTR1A (2.9%) and C22orf23 (1%). PR had more mutations than GR (median 65 vs 36, p=0.04). More PR than GR were HER2+ (5/14 vs 1/24, p=0.019) and/or TP53-mutated (5/10 vs 3/20, p=0.08) but similar proportions were PIK3CA-mutated. The correlation of diagnostic vs surgical variant allele frequencies was strong for the control (r=0.75) and treated (r=0.89) groups (for treated GR r=0.83; for treated PR r=0.65). In the treated group there were fewer mutations at surgery vs diagnosis (p Conclusion This is the largest reported study of exome reproducibility in ER+ BC for mutation profiles based on core-cut biopsy. Multiple sub-clones are identifiable in ER+ primary BC. In c.20% tumours, a single core-cut does not allow inference of all sub-clonal populations, probably due to spatial heterogeneity. TP53 mutations but not PIK3CA mutations are associated with PR. Large numbers of BC will be needed to identify any associations of lower frequency mutations with resistance. A trend to fewer mutations after just 2 weeks AI needs confirmation. Citation Format: Pascal Gellert, Corrinne V Segal, Qiong Gao, Tiandao Li, Christopher A Miller, Elaine Mardis, Lesley-Ann Martin, Christopher Holcombe, Anthony Skene, Judith Bliss, John Robertson, Ian Smith, Mitch Dowsett, POETIC Trial Management Group and Trialists. Exome sequencing of post-menopausal ER+ breast cancer (BC) treated pre-surgically with aromatase inhibitors (AIs) in the POETIC trial (CRUK/07/015) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-04.

The importance of early detection of lymphoedema by arm volume measurements before surgery and repeated measurements after surgery in women undergoing axillary node clearance (ANC) in order to enable early intervention is recognised. A prospective multi-centre study was performed which studied the difference between multi-frequency bioimpedance electrical analysis (BIS) and perometer arm measurement in predicting the development of lymphoedema. Women undergoing ANC underwent pre-operative and regular post-operative measurements of arm volume by both methods. The primary endpoint is the incidence of lymphoedema (≥10 % arm volume increase compared to contralateral arm by perometer) at 2 and 5 years after ANC. The threshold for intervention in lymphoedema was also assessed. Out of 964 patients recruited, 612 had minimum 6 months follow-up data. Using 1-month post-operative measurements as baseline, perometer detected 31 patients with lymphoedema by 6 months (BIS detected 53). By 6 months, 89 % of those with no lymphoedema reported at least one symptom. There was moderate correlation between perometer and BIS at 3 months (r = 0.40) and 6 months (r = 0.60), with a sensitivity of 73 % and specificity of 84 %. Univariate and multivariate analyses revealed a threshold for early intervention of ≥5 to

IMPORTANCE: Current treatment cures most cases of early-stage, primary breast cancer. However, better techniques are required to identify which patients are at risk of relapse. OBJECTIVE: To assess the clinical validity of molecular relapse detection with circulating tumor DNA (ctDNA) analysis in early-stage breast cancer. DESIGN, SETTING, AND PARTICIPANTS: This prospective, multicenter, sample collection, validation study conducted at 5 United Kingdom medical centers from November 24, 2011, to October 18, 2016, assessed patients with early-stage breast cancer irrespective of hormone receptor and ERBB2 (formerly HER2 or HER2/neu) status who were receiving neoadjuvant chemotherapy followed by surgery or surgery before adjuvant chemotherapy. The study recruited 170 women, with mutations identified in 101 patients forming the main cohort. Secondary analyses were conducted on a combined cohort of 144 patients, including 43 patients previously analyzed in a proof of principle study. INTERVENTIONS: Primary tumor was sequenced to identify somatic mutations, and personalized tumor-specific digital polymerase chain reaction assays were used to monitor these mutations in serial plasma samples taken every 3 months for the first year of follow-up and subsequently every 6 months. MAIN OUTCOMES AND MEASURES: The primary end point was relapse-free survival analyzed with Cox proportional hazards regression models. RESULTS: In the main cohort of 101 female patients (mean [SD] age, 54 [11] years) with a median follow-up of 35.5 months (interquartile range, 27.9-43.0 months), detection of ctDNA during follow-up was associated with relapse (hazard ratio, 25.2; 95% CI, 6.7-95.6; P

Introduction Women who undergo axillary surgery are at risk of developing lymphoedema. Early detection is recommended by measuring arm volume from a baseline before surgery to enable early intervention. The optimal measurement method to enable early detection and time to intervention are unclear. This prospective multi-centre study compares multi-frequency bioimpedance spectroscopy (BIS, ImpediMed) with the validated perometer method to determine which test is more sensitive for detecting the optimal threshold to prevent lymphoedema. Methods Participants (N = 960) undergoing axillary clearance at 9 UK centres have pre-operative and regular arm volume measurements post-surgery (1, 3, 6, 9 & 12 months, then 6 monthly), by the validated arm perometry compared with BIS (L-Dex) measurements as well as self-reported symptoms questionnaire. Change in arm volume was calculated using relative arm volume change (RAVC). The predictors of lymphoedema development and optimal method were assessed. Results Currently 612 patients, median age 55 (range 24 to 90) years, have 6 month follow-up data and 327 have 18 month follow-up data. Seventy six percent were ER positive and received endocrine therapy, 84% percent received radiotherapy and 67% received chemotherapy in addition to surgery. Lymphoedema by 18 months was detected in 19% (n=79) of women by perometry (≥10% RAVC) and a change in L-Dex of 10 was observed in 31% of women. A moderate correlation between perometer and BIS at 3 months (r=0.40) and 6 months (r=0.60), with a sensitivity of 73% and specificity of 84% was found. Univariate analysis revealed a threshold for early intervention to prevent lymphoedema was RAVC ≥5%- Conclusions The optimal threshold for early intervention to prevent progression to lymphoedema is ≥5%- (Funded by NIHR Programme Grant). Citation Format: Nigel J Bundred, Charlotte Stockton, Katie Riches, Linda Ashcroft, Abigail Evans, Anthony Skene, Maria Bramley, Tracey Hodgkiss, Arnie Purushotham, Vaughan Keeley, BEA Investigators. Optimal method of detection and threshold for early intervention to prevent lymphoedema: A multi-centre prospective study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-07.

Background Gene expression is widely used for the characterisation of breast cancers. Variability due to tissue heterogeneity or measurement error or systematic change due to peri-surgical procedures can affect measurements but is poorly documented. We studied the variability of global gene expression between core-cuts of primary ER+ breast cancers and the impact of delays to tissue stabilisation due to sample X-ray and of diagnostic core cutting. Methods Twenty-six paired core-cuts were taken immediately after tumour excision and up to 90 minutes delay due to sample X-ray; 57 paired core-cuts were taken at diagnosis and 2 weeks later at surgical excision. Whole genome expression analysis was conducted on extracted RNA. Correlations and differences were assessed between the expression of individual genes, gene sets/signatures and intrinsic subtypes. Results Twenty-three and 56 sample pairs, respectively, were suitable for analysis. The range of correlations for both sample sets were similar with the majority being >0.97 in both. Correlations between pairs for 18 commonly studied genes were also similar between the studies and mainly with Pearson correlation coefficients >0.6 except for a small number of genes, which had a narrow-dynamic range (e.g. MKI67, SNAI2). There was no systematic difference in intrinsic subtyping between the first and second sample of either set but there was c.15 % discordance between the subtype assignments between the pairs, mainly where the subtyping of individual samples was less certain. Increases in the expression of several stress/early-response genes (e.g. FOS, FOSB, JUN) were found in both studies and confirmed findings in earlier smaller studies. Increased expression of IL6, IGFBP2 and MYC (by 17 %, 14 % and 44 %, respectively) occurred between the samples taken 2 weeks apart and again confirmed findings from an earlier study. Conclusions There is generally good correlation in gene expression between pairs of core-cuts except where genes have a narrow dynamic range. Similar correlation coefficients to the average gene expression profiles of intrinsic subtype, particularly LumA and LumB, can lead to discordances between assigned subtypes. Substantial changes in expression of early-response genes occur within an hour after surgery and in IL6, IGFB2 and MYC as a result of diagnostic core-cut biopsy. Trial registration Trial number CRUK/07/015. Study start date September 2008. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0696-2) contains supplementary material, which is available to authorized users.

Background. Detection of circulating tumor DNA (ctDNA) after treatment of early stage breast cancer may identify molecular residual disease. In a prior proof-of-principle study we demonstrated that detection of ctDNA predicted relapse with high accuracy (Garcia-Murillas et al Science Trans Med 2015). We conducted an independent, prospective, multi-centre validation study. Methods. In this validation study, a cohort of 170 early stage breast cancer patients were recruited from five hospitals into two prospective sample collection studies. Patients were scheduled to receive standard chemotherapy, surgery +/- radiotherapy, adjuvant endocrine therapy and HER2 antibodies as appropriate. Plasma samples were collected for ctDNA analysis at baseline, post-surgery, three monthly for the first year of follow-up, and six monthly thereafter and shipped to a central lab for processing. Using previously established criteria, tumor was sequenced to identify somatic mutations that were tracked by digital PCR in DNA extracted from 4mls of plasma at all available time points. Buffy coat DNA was analysed at all time-points to control for clonal haematopoesis of indeterminate potential (CHIP) detection. The primary endpoint was to compare invasive disease free survival between patients with and without detection of ctDNA after treatment. A combined analysis of this validation study, and the prior proof-of-principle study, was also conducted to analyse secondary endpoints. Results. After tumor sequencing, 101 patients from the validation study had at least one mutation to track. At median 35.5 months follow-up, ctDNA was detected in plasma of 15.8% (16/101) patients. Detection of ctDNA strongly predicted relapse, hazard ratio 24.5 (95% CI 6.5 to 93.2, P In the combined analysis (N=144), lead-time between ctDNA detection and relapse was 10.7 months (95% CI 7.7-17.0). Six patients had a clinical relapse that was not detected by ctDNA prior to relapse. These patients had a distinct pattern of oligo-metastatic relapse, 3 patients with brain-only metastases (P=0.0068), 1 ovarian oligo-metastasis and 2 local disease recurrence. The level of ctDNA in baseline plasma, prior to treatment, was associated with tumor subtype, highest in triple negative breast cancer (P=0.0036). Conclusion. Detection of ctDNA after treatment is associated with a high risk of future relapse in early-stage breast cancer. Prospective studies are required to assess the potential of molecular residual disease detection to guide adjuvant therapy. Citation Format: Turner N, Garcia-Murillas I, Chopra N, Comino-Mendez I, Beaney M, Tovey H, Cutts R, Swift C, Kriplani D, Afentakis M, Hrebien S, Walsh G, Johnston S, Ring A, Russell S, Evans A, Skene A, Wheatley D, Dowsett M, Smith I. Molecular residual disease detection with circulating tumor DNA analysis predicts relapse in patients with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-01.

This abstract was withdrawn by the authors. Citation Format: Bundred NJ, Foden P, Riches K, Morris J, Evans A, Todd C, Bramley M, Skene A, Purushotham A, Keeley V. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-42.

Introduction: The substantial majority of breast cancers present with early-stage disease, although micro-metastatic disease may be established at the time of diagnosis and may ultimately result in metastatic disease recurrence. Prior studies have shown that analysis of circulating tumour DNA (ctDNA) can detect micro-scopic minimal residual disease (MRD) serving as biomarker to anticipate future cancer recurrence, although sensitivity of current assays is limited. We present a novel molecular fingerprint ctDNA assay that exploits tumour whole genome sequencing (WGS) to develop highly personalised assays. Methods: WGS was conducted on paired tumor and germline DNA from 11 early breast cancer patients to identify tumour specific small indels (SIs) to track in plasma as individual molecular fingerprints. Patient-specific amplicon-based panels were designed and validated to identify somatic reporters with no sequencing background. DNA was extracted from plasma samples taken at pre-surgery (baseline), post-surgery, and every 3 months until 1 year and every 6 months thereafter, and sequenced to 100,000X. Results: Molecular fingerprint assays were demonstrated to detect less than a single cancer genome, with undetectable background in control samples. Using plasma samples from patients with early stage breast cancer, we detected ctDNA in 80% of the baseline plasma time-points with 100% specificity (95% confidence limits (CI) 98.7%-100%). ctDNA was detected in all patients prior to relapse, with an observed lead-time of 15.5 months, substantially improved compared with previous techniques, detecting residual disease that was not detectable by tracking mutations using digital PCR. Conclusions: This proof-of-principle study demonstrates that tumor-specific molecular fingerprints combined with ultra-deep plasma DNA sequencing have the potential to transform MRD detection. Identifying those patients who have undetectable recurrence would pave the way for identifying who is cured by surgery alone and who requires further therapy. Citation Format: Iñaki Comino-Mendez, Ros Cutts, Isaac García-Murillas, Neha Chopra, Maria Afentakis, Abigail Evans, Duncan Wheatley, Anthony Skene, Simon Russell, Mohini Varughese, Mitch Dowsett, Ian E. Smith, Nick Turner. Molecular fingerprint sequencing for minimal residual disease detection in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3608.

Background: AKT is an important intracellular control point through which Type 1 growth factors and IGFR signal. Mutations in PIK3CA, AKT and PTEN are prevalent in estrogen receptor positive (ER+) breast cancer (BC) and have been implicated in resistance to endocrine therapies. AZD5363 is an inhibitor of AKT 1, 2 and 3 currently in Phase 2 trials for BC and other solid cancers. Design: The study examined whether AZD5363 impacts on key biomarkers within the AKT pathway and their subsequent effects on Ki67, a marker of tumor proliferation. STAKT is a multi-center, two-stage, double blind, randomized, placebo controlled, biomarker 'window-of-opportunity' trial in women with newly diagnosed, previously untreated ER+ BC who were deemed would require chemotherapy as part of their primary treatment regimen. Stage 1 assessed AZD5363 at a dose of 480mg bd p.o. versus matching placebo. Up to 30 patients per arm were permitted, to allow 12 subjects per arm with evaluable paired biopsies - obtained at baseline, and after 4.5 days of AZD5363 / placebo. Primary endpoint markers were pPRAS40, pGSK3β and Ki67 assessed by immunohistochemistry. pPRAS40 and pGSK3β were assessed by H-scores and measured separately for cytoplasmic (cyto), nuclear (nuc) and total (cyto+nuc) staining. Ki67 was assessed as % positive staining of 500 tumor nuclei. Laboratory staff were blinded to treatment arm and whether the biopsies were taken before or after AZD5363/placebo. Changes in marker expression (both absolute and %) between biopsies were calculated, and compared between the two groups. An ANOVA test was applied for normally distributed data and Wilcoxon Mann-Whitney used if not normally distributed. Results: 28/36 patients were evaluable with patient & tumor characteristics as follows: 17 received AZD5363 and 11 placebo; the median ages were 48 & 49 years respectively. 27 patients were Caucasian and 1 African-American. Tumors were all ER+. For HER2 status 8 were positive & 9 negative in the AZD5363 treated group compared to 2 & 9 respectively in the placebo group. For pPRAS40 and pGSK3β cyto was the predominant staining while for Ki67 staining was nuclear. Changes in each marker with associated p-values are shown in the table. MarkerType of change vs baselineDegree of change in AZD5363 arm (n=17)p-value versus placebo arm (n=11)pPRAS40 (H-score)TotalAbsolute-83.8 Conclusions• AZD5363 for 4.5 days caused highly significant falls in pGSK3β and pPRAS40, key markers of AKT pathway activation • AZD53643 also caused a significant decline in Ki67 even after only 4.5 days of drug. This is one of the shortest 'window'-studies to report such an early effect on proliferation. • Placebo controlled 'window' studies of this short duration can provide important evidence of the therapeutic potential early in a drug's development. Citation Format: Robertson JFR, Coleman RE, Cheung KL, Evans A, Holcombe C, Skene A, Rea D, Ahmed S, Jahan A, Kelly S, Horgan K, Rauchhaus P, Littleford R, Foxley A, Lindemann JPO, Pass M, Rugman P, Deb R, Finlay P, Gee JMW. AZD5363, an AKT inhibitor, significantly inhibits key biomarkers of the AKT pathway and Ki67, in a randomized, placebo, controlled study (STAKT) in human breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-06.

This abstract was withdrawn by the authors.